Methods and kits for diagnosis, prognosis or monitoring of Epstein-Barr virus (EBV)-associated cancer

ABSTRACT

Disclosed is a non-invasive method for diagnosis, prognosis or monitoring of Epstein-Barr virus (EBV)-associated cancer by detecting and/or quantifying EBV associated nucleic acid fragments in a urine sample from an individual. Kits for diagnosis, prognosis or monitoring of cancer are also disclosed.

FIELD OF THE INVENTION

The present invention generally relates to methods and kits fordiagnosis, prognosis or monitoring of a cancer in an individual bydetecting and/or quantifying tumor-associated DNA sequences. Morespecifically, this invention relates to methods and kits for diagnosis,prognosis or monitoring of an Epstein-Barr virus (EBV)-associated cancerby detecting and/or quantifying an EBV DNA sequence in urine.

BACKGROUND OF THE INVENTION

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that wasfirst discovered in association with the African form of Burkitt'slymphoma (BL). Subsequently, EBV infection is also found to be stronglyassociated with nasopharyngeal carcinoma (NPC).

It has been shown that the EBV genome can be detected in almost all NPCtissues collected in Southern China (Chen et al., Intervirology.36(2):91-8 (1993); Dickens et al., J Clin Pathol. 45(5):396-7 (1992)).Consistent with these results, we have demonstrated that EBV DNA can bedetected in the plasma of 96% of NPC patients but only 7% of healthysubjects at a much lower concentration (Lo et al., Cancer Res.59(6):1188-91 (1999)). Moreover, the level of plasma EBV DNA, measuredeither before (Lo et al., Cancer Res. 60(24):6878-81 (2000)) or aftertreatment (Chan et al., J Natl Cancer Inst. 94(21):1614-9 (2002)), isvaluable for predicting the overall and disease-free survival.Similarly, circulating EBV DNA is also useful for the detection andmonitoring of other EBV-associated malignancies, for example, lymphoma(Lei et al., Br J Haematol. 111(1):239-46 (2000)) and gastric carcinoma(Lo et al., Clin Cancer Res. 7(7):1856-9 (2001)).

Although this plasma test is useful for the detection of NPC and is arelatively risk free procedure, it still causes pain and anxiety inpatients. However, detection and/or quantification of short EBVassociated nucleic acid sequences in a urine sample, as well as usesthereof, have not been disclosed in the art. Accordingly, there is aneed for develop an alternative test for diagnosis, prognosis, and/ormonitoring of EBV-associated cancers.

SUMMARY OF THE INVENTION

We surprisingly find that the circulating cell-free Epstein-Barr virus(EBV) DNA can pass through the kidney barrier and be detected in urineas a tumor marker for NPC and other EBV-associated malignancies.

According to a first aspect of the present invention, there is provideda method for diagnosis of an EBV-associated cancer in an individual. Themethod comprises:

(a) obtaining a urine sample from the individual; and

(b) detecting an EBV associated nucleic acid sequence in the urinesample,

wherein the presence of the EBV associated nucleic acid sequence in theurine sample indicates that the individual is suffering from anEBV-associated cancer.

According to a second aspect of the present invention, there is provideda method for prognosis of an EBV-associated cancer in an individual. Themethod comprises:

(a) obtaining a urine sample from the individual; and

(b) detecting an EBV associated nucleic acid sequence in the urinesample,

wherein the presence of the EBV associated nucleic acid sequence in theurine sample indicates a poor prognosis of the EBV-associated cancer.

According to a third aspect of the present invention, there is provideda method for monitoring an EBV-associated cancer in an individual. Themethod comprises:

(a) obtaining urine samples from the individual at different timepoints; and

(b) detecting and/or quantifying an EBV associated nucleic acid sequencein the urine samples,

wherein the presence or an increased level of the EBV associated nucleicacid sequence in the urine samples indicates the progression of theEBV-associated cancer, and the absence or a decreased level of the EBVassociated nucleic acid sequence in the urine samples indicates theregression of the EBV-associated cancer.

In a specific embodiment of the method of the invention, step (a) isperformed during the time course of a treatment for the EBV-associatedcancer, and the absence or a decreased level of the EBV associatednucleic acid sequence in the urine samples indicates the effectivenessof the treatment.

In preferred embodiments of the present invention, the methods describedabove further comprise the step of analyzing the size of the EBVassociated nucleic acid sequence in the urine samples to eliminate falsepositive cases. In these cases, the EBV associated nucleic acid sequencedetected and/or quantified in the urine samples may not be derived fromthe circulating EBV associated nucleic acid fragments and is relativelyintact.

In preferred embodiments of the present invention, the EBV associatednucleic acid sequence described above is derived from at least one DNAfragment of the BamHI-W region of the EBV genome or at least one RNAtranscript thereof. Preferably, the DNA fragment or the RNA transcriptis less than 180 nucleotides.

According to a fourth aspect of the present invention, there is provideda kit for diagnosis or prognosis of an EBV-associated cancer in anindividual. The kit comprises:

a) a first unit for extracting nucleic acid from a urine sample from theindividual; and

b) a second unit for detecting an EBV associated nucleic acid sequencein the extracted nucleic acid, wherein the second unit comprises atleast one pair of primers for amplifying at least one fragment of theBamHI-W region of the EBV genome.

According to a fifth aspect of the present invention, there is provideda kit for monitoring an EBV-associated cancer in an individual. The kitcomprises:

a) a plurality of first units for extracting a nucleic acid from a urinesample from the individual; and

b) a plurality of second units for detecting and/or quantifying an EBVassociated nucleic acid sequence in the extracted nucleic acid, whereinthe second unit comprises at least one pair of primers for amplifying atleast one fragment of the BamHI-W region of the EBV genome.

In one preferred embodiment, the kit according to the present inventionfurther comprises a device for obtaining a urine sample from anindividual.

In some embodiments of the present invention, the EBV associated nucleicacid described above is DNA. In other embodiments, it is RNA.

In some preferred embodiments of the present invention, the EBVassociated cancer is a nasopharyngeal carcinoma (NPC), anatural-killer-cell lymphoma (NK-lymphoma) or a gastric carcinoma. Inmore preferred embodiments, the EBV-associated cancer is anasopharyngeal carcinoma.

Advantages of the present invention will become more apparent to one ofordinary skill in the art from the following description of thepreferred embodiments of the present invention that have been shown anddescribed by way of illustration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the plasma EBV DNA concentrations in NPC patients accordingto the detectability of urine EBV DNA; and

FIG. 2 shows correlation between the plasma EBV DNA and the urine EBVDNA levels in NPC patients with detectable urine EBV DNA, in which (a):the urine EBV DNA concentrations are expressed in copies/mL of urine and(b): the urine EBV DNA concentrations are expressed in copies/mmol ofcreatinine in the urine.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs.

As described above, the methods of the present invention can be used todiagnose or prognosticate an EBV-associated cancer, such asnasopharyngeal carcinoma (NPC). In these methods, a urine sample isfirst obtained from an individual and the detection and/orquantification of an EBV DNA sequence in the urine sample is thenperformed.

A method for monitoring an EBV-associated cancer in an individual isalso disclosed. The method comprises steps of (a) obtaining urinesamples from the individual at different time points; and (b) detectingand/or quantifying an EBV DNA sequence in the urine samples. Adifference in the levels of the EBV DNA sequence in the urine samples isused as an indication of the development of the EBV-associated cancer.For example, the presence or an increased level of the EBV DNA sequencein the urine samples indicates the progression of the EBV-associatedcancer, and the absence or a decreased level of the EBV DNA sequence inthe urine samples indicates the regression of the EBV-associated cancer.This method is particularly useful for assessing the effectiveness of atreatment for an EBV-associated cancer.

Though the step of obtaining a urine sample from the individual isincluded in the methods described above, it will be appreciated by thoseskilled in the art that this step is not necessary in some cases, inwhich the urine sample has been obtained in advance, for example, at asampling center.

In preferred embodiments, the detection or quantification of an EBV DNAsequence in the urine sample comprises the steps of (1) extracting DNAfrom the urine sample; (2) amplifying the EBV DNA sequence from theextracted DNA; and (3) detecting and/or quantifying the amplified EBVDNA sequence.

As used herein, the expression “an EBV DNA sequence” refers to afragment of the Epstein-Barr virus (EBV) genome (Genbank accessionnumber AJ507799) and typically is a product of the genome DNAdegradation. In particular, the EBV DNA sequence according to thepresent invention comprises at least one fragment of the BamHI-W regionof the EBV genome. The BamHI-W region used herein refers to a repeatingBamHI-W restriction fragment of the EBV genome as disclosed in Jones etal., Nucleic Acids Res. 11:3919-3937 (1983).

It is understood that a longer DNA sequence will pass through the kidneybarrier at a lower rate. Thus, in order to increase the sensitivity ofthe detection, the size of the selected EBV DNA sequence according tothe present invention is no more than 180 bp. Preferably, the size ofthe target DNA sequence according to the present invention is no morethan 76 bp.

As used herein, the term “amplifying” or “amplification” means theproduction of additional copies of the EBV DNA sequence and is generallycarried out using polymerase chain reaction or other technologies wellknown in the art. With PCR, it is possible to amplify a single copy ofan EBV DNA sequence to a level that can be detected by several differentmethodologies.

In a preferred embodiment of the invention, the step (2) and step (3)are carried out by real-time quantitative PCR (Q-PCR). As used herein,the term “real-time quantitative PCR” or “Q-PCR” refers to a methodbased on the continuous optical monitoring of the progress of afluorogenic PCR reaction. In this system, in addition to a pair ofamplification primers used in conventional PCR, a dual-labeledfluorogenic hybridization probe is also included. One fluorescent dyeserves as a reporter (e.g. FAM), and its emission spectra is quenched bya second fluorescent dye (e.g. TAMRA). During the extension phase ofPCR, the 5′ to 3′ exonuclease activity of Taq DNA polymerase cleaves thereporter from the probe, thus releasing it from the quencher andresulting in an increase in fluorescence. The fluorescence is then usedto monitor the amplification process and determine the amount of theoriginal template DNA.

It is found in our research that healthy subjects may occasionally carryEBV DNA in urine. In order to eliminate these false positive cases, themethods of the present invention further include a step of analyzing thesize of the EBV DNA sequence present in the urine sample.

In some embodiments, the analyzing of the size of the EBV DNA sequenceis carried out by two or more PCR assays or Q-PCR assays. These assaysuse amplicons of different sizes. Because a longer DNA sequence passesthrough the kidney barrier at a lower rate, a much lower concentrationof the final amplification product will be obtained after the sameamplification cycles when larger amplicons are used. If two or more PCRassays or Q-PCR assays targeting an EBV DNA sequence using amplicons ofdifferent sizes (e.g., 59 bp and 76 bp) give similar amounts of thefinal amplification products, the EBV DNA sequence present in the urinemay not come from the circulating EBV DNA passed through the kidneybarrier. Elimination of these cases renders the methods of the presentinvention higher reliability for the diagnosis, prognosis or monitoringof NPC and other EBV-associated malignancies.

Alternatively, we can conveniently analyze whether an EBV genomefragment of more than 180 bp is present in the urine sample. As a DNAfragment of more than 180 bp is difficult to pass through the kidneybarrier, the presence of an EBV genome fragment of more than 180 bp inthe urine sample can be used to exclude the individual from a patientwith an EBV-associated cancer.

As described previously, the plasma EBV DNA concentration can be used todiagnose, prognosticate or monitor NPC and other EBV-associatedmalignancies. It is disclosed in the present invention that there is agood correlation between the plasma and the urine EBV DNAconcentrations, i.e. the plasma EBV DNA concentration can be determinedby quantifying the urine EBV DNA concentration. Therefore, the assay forthe concentration of an EBV DNA sequence in urine is an excellentalternative to the assay in plasma for diagnosing, prognosticating ormonitoring NPC and other EBV-associated malignancies.

According to the present invention, the presence of an EBV DNA sequencein a urine sample from an individual can be used as an indication of anEBV-associated cancer, including the stage of the EBV-associated cancer.According to the present invention, the presence of an EBV DNA sequencein a urine sample from an individual can also be used as a poorprognostic factor of an EBV-associated cancer, and for the prediction ofthe likelihood of overall survival or recurrence for the individual.

When the level of an EBV sequence in the urine of an individual with anEBV-associated cancer is followed for a period of time, the trend of thelevel reflects the development of the cancer. Generally, an increasedlevel is an indication of the progression of the cancer. The trend ofthe level of an EBV sequence in the urine can also be used to assess theeffectiveness of a treatment for the EBV-associated cancer. The absenceor a decreased level of the EBV DNA sequence in the urine sample is agood indication of the effectiveness of the treatment.

EBV DNA is named above as an illustrative example of EBV associatednucleic acid. It is well-known to those of skill in the art that thereare other types of nucleic acids, e.g. RNA which are associated withEBV. Accordingly, in certain embodiments, the methods for diagnosis,prognosis and monitoring of EBV-associated cancers comprising detectingEBV associated RNA, such as Epstein-Barr encoded small RNA (EBER) in aurine sample of a human subject.

The present invention also features a kit for diagnosis or prognosis ofan EBV-associated cancer in an individual. This kit comprises a) a firstunit for extracting DNA from a urine sample; and b) a second unit fordetecting and/or quantifying an EBV DNA sequence in the extracted DNA,wherein the second unit comprises at least one pair of primers foramplifying at least one fragment of the BamHI-W region of the EBVgenome. The present invention also features a kit for monitoring anEBV-associated cancer in an individual. This kit comprises a) aplurality of first units for extracting DNA from a urine sample; and b)a plurality of second units for detecting and/or quantifying an EBV DNAsequence in the extracted DNA, wherein the second unit comprises atleast one pair of primers for amplifying at least one fragment of theBamHI-W region of the EBV genome.

In preferred embodiments, the kits according to the present inventionfurther comprise a device for obtaining a urine sample from anindividual. If desired, the kits may also comprise instructions forusing the kits. Typically, the second unit of the kits further comprisesa probe for a Q-PCR assay. In order to eliminate the false positivecases mentioned above, the kits preferably comprise primers foramplicons of different sizes. For example, the kits include a pair ofprimers for amplifying an EBV DNA sequence of 180 bp and another pair ofprimers for amplifying an EBV DNA sequence of 76 bp.

The kit according to the present invention for monitoring anEBV-associated cancer is particularly useful for assessing theeffectiveness of a treatment for an EBV-associated cancer, as theabsence or a decreased level of the EBV DNA sequence in the urine samplecan be used as an indication of the effectiveness of the treatment.

It is apparent to those skilled in the art that the kits according tothe present invention can also be used to detect and/or quantify an EBVRNA sequence in a urine sample by using a reverse transcriptasepolymerase chain reaction (RT-PCR) assay or a real-time quantitativereverse transcriptase polymerase chain reaction (Q-RT-PCR) assay, forthe diagnosis, prognosis or monitoring of an EBV-associated cancer in anindividual.

The following examples are provided by way of illustration only and notby way of limitation.

EXAMPLES

44 NPC patients, as well as 70 healthy control subjects, were recruited.DNA was first extracted from urine and plasma samples from the subjects,and the extracted DNA was then amplified for EBV DNA using quantitativereal-time PCR targeting the BamHI-W region (Lo 1999 et al.) of the EBVgenome, as described below.

Extraction of the DNA Fragments from Urine and Plasma

Ten milliliters of urine were collected from each study subject into anEDTA-containing tube. The urine samples were centrifuged at 1,600 g for10 minutes at 4° C. The supernatant was filtered with a 0.45 μm filterto remove any remaining cells. DNA was extracted from the urine sampleusing the Wizard Plus Miniprep DNA Purification Kit (Promega, Madison,Wis.). The filtered supernatant was mixed thoroughly with 15 mL 6Mguanidine isothiocyanate (GITC). One milliliter of resin suspension ofthe DNA purification kit was added to each sample and the mixture wasmixed thoroughly on a roller-mixer for 2 hours at room temperature. Theurine-resin mixture was then passed through the minicolumn provided inthe kit using a 30 mL syringe. Two milliliters of wash solution werethen passed through the minicolumn. The remaining wash solution wascleared by a brief centrifugation. One hundred microliters H₂O wereadded to the minicolumn for the elution of DNA fragments.

The method for extracting DNA fragments from plasma is known in the art,e.g., the method by using a QIAamp Blood Kit (Qiagen, Hilden, Germany)described by Lo et al., Cancer Res. 60(24):6878-81 (2000). In brief,plasma samples were harvested from the patients according to establishedprotocols in the art. The samples were stored at −20° C. until furtherprocessing. DNA from plasma samples was extracted using a QIAamp BloodKit (Qiagen, Hilden, Germany) using the “blood and body fluid protocol”as recommended by the manufacturer. A total of 400-800 μl of the plasmasamples were used for DNA extraction per column. The exact amount wasdocumented for the calculation of the target DNA concentration. A finalelution volume of 50 μl was used to elute the DNA from the extractioncolumn.

Detection and Quantification of the EBV DNA Fragments

The concentration of urine EBV DNA was determined by two real-time PCRassays both targeting the BamHI-W region of the EBV genome. The ampliconsizes for the two PCR assays were 76 bp and 59 bp. The primers used forthe 76 bp assay were 5′-CCCAACACTCCACCACACC-3′ (SEQ ID NO. 1) and5′-TCTTAGGAGCTGTCCGAGGG-3′ (SEQ ID No. 2). The primers used for the 59bp assay were 5′-CCCAGGCACACACTACACACA-3′ (SEQ ID No. 3) and5′-TCTTAGGAGCTGTCCGAGGG-3′ (SEQ ID No. 4). The fluorescent probes usedfor the 76 bp and 59 bp assays were5′-(FAM)-CACACACTACACACACCCACCCGTCTC-(TAMRA)-3′ (SEQ ID No. 5) and5′-(FAM)-CACCCGTCTCAGGG-(MGB)-3′ (SEQ ID No. 6), respectively. The PCRreactions were set up in a reaction volume of 50 μL. Each reactioncontained 5 μL of 10× buffer A; 300 nM of each of the amplificationprimers; 25 nM of the corresponding fluorescent probe; 4 mM MgCl₂; 200μM each of dATP, dCTP, and dGTP, 400 μM dUTP; 1.25 units of AmpliTaqGold; and 0.5 unit of AmpErase uracil N-glycosylase. Ten microliters ofextracted urine DNA were used as a template. An identical thermalprofile was used for both PCR systems and was 50° C. for 2 min, 95° C.for 10 min, followed by 40 cycles of 95° C. for 15 s and 56° C. for 1min. A calibration curve was run in parallel with each analysis, usingDNA extracted from the EBV-positive cell line, Namalwa, as a standard.Namalwa is a diploid cell line that contains two integrated EBV genomesper cell. Each sample was analyzed in duplicate and the mean value wastaken for analysis.

The quantity of the urine EBV DNA can be expressed as a concentrationper unit volume of urine, for example copies/mL urine, or expressed as aquantity per the amount of another substance in the urine sample tocorrect for the concentration of the urine, for example copies/mmol ofcreatinine.

The Presence or the Level of EBV in the Urine Sample Being a UsefulClinical Marker for NPC

EBV DNA was detectable in 10 (23%) and 22 (50%) urine samples from NPCpatients using the 76 bp and 59 bp assays, respectively (Table 1). Thehigher detection rate for the 59 bp assay indicates that shorter DNAmolecules are more readily filtered through the kidney barrier and bedetected in the urine. Consistent with our previous results, the EBV DNAcan be detected in almost all of the plasma samples from NPC patients.As shown in Table 1, 41 (93%) plasma samples from NPC patients can bedetected using either the 59 bp assay or the 76 bp assay. A furtherreduction in the size of the target fragment may further increase thesensitivity of EBV associated nucleic acid detection.

TABLE 1 The detection rates of urine EBV DNA in NPC patients using the59 bp and 76 bp assays Plasma EBV DNA positive EBV DNA negative 59 bpEBV Urine positive 22 0 DNA assay Urine negative 19 3 76 bp EBV Urinepositive 10 0 DNA assay Urine negative 31 3

70 healthy subjects were also analyzed as controls. EBV DNA wasdetectable in the plasma of 3 (4%) subjects using the 76 bp assay (Table2). Their plasma EBV DNA concentrations were 13 copies/mL, 17 copies/mLand 22 copies/mL. These levels were relatively low when compared withthe plasma EBV DNA levels of the 44 NPC patients (median: 950copies/mL). EBV DNA was not detectable in the urine of these threecontrol subjects.

TABLE 2 The detection rates of urine EBV DNA in NPC patients and healthycontrol subjects using the 59 bp EBV DNA assay NPC patients Healthycontrol subjects Urine EBV DNA Positive 22 2 Negative 22 68

Elimination of False Positive Cases

Surprisingly, EBV DNA was detectable in the urine of two healthysubjects with no detectable plasma EBV DNA at the concentration of 227copies/mL and 1,280,000 copies/mL.

These two urine samples were further analyzed with 2 real-time PCRassays targeting the BamHI-W region of the EBV genome with ampliconsizes of 76 bp and 180 bp. The urine EBV DNA concentrations for thesetwo subjects were relatively constant using the two assays. For theformer subject, the urine EBV DNA concentrations were 333 copies/mL and243 copies/mL using the 76 bp and 180 bp assays, respectively. For thelatter subject, the urine EBV DNA were 1,220,000 copies/mL and 1,073,000copies/mL using the 76 bp and 180 bp assays, respectively. In contrast,for the NPC patients with detectable urine EBV DNA using both the 59 bpand the 76 bp assays, the median drop in concentration was 93.5%. UrineEBV DNA was not detectable in the urine of the 44 NPC patients using the180 bp assays. The relatively constant urine EBV DNA concentrations ofthe two healthy subjects using the two different-sized PCR assayssuggests that the EBV DNA molecules detected in their urine wererelatively intact when compared with the urine EBV DNA detected in theurine of the NPC patients. One possible explanation would be thepresence of intact viral particles in the urine of the two controlsubjects which may be resulted from active viral replication.Diagnostically, the size of urine EBV DNA can be included in theanalysis to increase the specificity of urine EBV DNA analysis.

Correlation Between Plasma and Urine EBV DNA Concentrations

The plasma EBV DNA concentrations of the NPC patients were analyzedusing the 76 bp real-time PCR assay. The median plasma EBV DNAconcentrations for the patients with detectable and undetectable urineEBV DNA were 2054 copies/mL and 511 copies/mL, respectively (p<0.0001,Mann-Whitney test). The plasma EBV DNA concentrations were statisticallysignificantly higher in patients with detectable urine EBV DNA. Theresults are shown in FIG. 1.

For those patients with detectable urine EBV DNA, we have investigatedif there is any correlation between the plasma and urine EBV DNAconcentrations. There was a significant positive correlation between theplasma and urine EBV DNA concentrations (r=0.684, p<0.0001, Spearmancorrelation; FIG. 2 a). As the urine concentration of EBV DNA can beaffected by the hydration status of a patient, we have corrected theurine concentration of EBV DNA with the urine concentration ofcreatinine and expressed the urine EBV DNA concentration in copies/mmolcreatinine. The positive correlation between the plasma and urine EBVDNA concentrations becomes more prominent when the urine EBV DNAconcentrations were corrected with the urine creatinine concentrations(r=0.748, p<0.0001, Spearman correlation; FIG. 2 b).

The Presence or the Level of EBV DNA in the Urine Sample Being a UsefulIndication of Prognosis

After a median follow up of 8 months, 3 of the 44 NPC patients haddeveloped clinical recurrence. All the 3 patients had detectable EBV DNAin urine before treatment. In contrast, none of the 22 patients withundetectable urine EBV DNA had developed clinical relapse. As patientswith detectable urine EBV DNA had a significantly higher plasma EBV DNAlevels, it is expected that urine EBV DNA can also be served as aprognostic marker for post-treatment survival, because it is known ahigh plasma EBV DNA level is a poor prognostic factor for diseaserecurrence after treatment. It can be expected the difference insurvival probability would become more prominent with the increase infollow up duration.

It should be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety.

REFERENCES

Chan, A T C, Lo, Y M D, Zee, B, Chan, L Y S, et al. (2002) PlasmaEpstein-Barr virus DNA and residual disease after radiotherapy forundifferentiated nasopharyngeal carcinoma. J Natl Cancer Inst94(21):1614-9.

Chen, C L, Wen, W N, Chen, J Y, Hsu, M M, et al. (1993) Detection ofEpstein-Barr virus genome in nasopharyngeal carcinoma by in situ DNAhybridization. Intervirology; 36(2):91-8.

Dickens, P, Srivastava, G, Loke, S L, Chan, C W, et al. (1992)Epstein-Barr virus DNA in nasopharyngeal carcinomas from Chinesepatients in Hong Kong. J Clin Pathol; 45(5):396-7.

Jones, M D and Griffin, B E. (1983) Clustered repeat sequences in thegenome of Epstein-Barr virus. Nucleic Acids Res; 11:3919-3937.

Lei, K I, Chan, L Y S, Chan, W Y, Johnson, P J, et al. (2000)Quantitative analysis of circulating cell-free Epstein-Barr virus (EBV)DNA levels in patients with EBV-associated lymphoid malignancies. Br JHaematol 111(1):239-46.

Lo, Y M D, Chan, A T C, Chan, L Y S, Leung, S F, et al. (2000) Molecularprognostication of nasopharyngeal carcinoma by quantitative analysis ofcirculating Epstein-Barr virus DNA. Cancer Res; 60(24):6878-81.

Lo, Y M D, Chan, L Y S, Lo, K W, Leung, S F, et al. (1999) Quantitativeanalysis of cell-free Epstein-Barr virus DNA in plasma of patients withnasopharyngeal carcinoma. Cancer Res;59(6):1188-91.

Lo, Y M D, Chan, W Y, Ng, E K, Chan, L Y, et al. (2001) CirculatingEpstein-Barr virus DNA in the serum of patients with gastric carcinoma.Clin Cancer Res;7(7):1856-9.

1. A method for diagnosis of an Epstein-Barr virus (EBV)-associatedcancer in an individual comprising: (a) obtaining a urine sample fromthe individual; and (b) detecting an EBV associated nucleic acidsequence in the urine sample, wherein the presence of the EBV sequencein the urine sample indicates that the individual is suffering from anEBV-associated cancer.
 2. The method according to claim 1, wherein step(b) comprises: (b1) extracting a nucleic acid from the urine sample;(b2) amplifying the EBV associated nucleic acid sequence from theextracted nucleic acid sample; and (b3) detecting the amplified EBVassociated nucleic acid sequence.
 3. The method according to claim 1,wherein the EBV associated nucleic acid is DNA.
 4. The method accordingto claim 1, wherein the EBV associated nucleic acid sequence is derivedfrom at least one fragment of the BamHI-W region of the EBV genome. 5.The method according to claim 4, wherein the at least one fragment isless than 180 nucleotides.
 6. The method according to claim 1, furthercomprising the step of analyzing whether an EBV genome fragment of morethan 180 nucleotides is present in the urine sample, wherein thepresence of an EBV genome fragment of more than 180 nucleotides in theurine sample indicates that the individual is not suffering from anEBV-associated cancer.
 7. The method according to claim 1, wherein twofragments of the BamHI-W region are quantified in step (b), wherein thetwo fragments are of different sizes and both are less than 180nucleotides, and wherein substantially equal levels of the two fragmentsin the urine sample indicates that the individual is not suffering froman EBV-associated cancer.
 8. The method according to claim 1, wherein apolymerase chain reaction (PCR) assay, a reverse transcriptasepolymerase chain reaction (RT-PCR) assay, a real-time quantitativepolymerase chain reaction (Q-PCR) assay, or a real-time quantitativereverse transcriptase polymerase chain reaction (Q-RT-PCR) assay isperformed in step (b).
 9. The method according to claim 8, wherein apair of primers used in the PCR or Q-PCR assay are selected from thegroup consisting of SEQ ID NO. 1 and SEQ ID NO. 2; and SEQ ID NO. 3 andSEQ ID NO.
 4. 10. The method according to claim 8, wherein a probe usedin the Q-PCR assay has a DNA sequence of SEQ ID NO. 5 or SEQ ID NO.6.11. The method according to claim 1, wherein the EBV-associated canceris a nasopharyngeal carcinoma (NPC), a natural-killer-cell lymphoma(NK-lymphoma) or a gastric carcinoma.
 12. A method for prognosis of anEBV-associated cancer in an individual comprising: (a) obtaining a urinesample from the individual; and (b) detecting an EBV associated nucleicacid sequence in the urine sample, wherein the presence of the EBVassociated nucleic acid sequence in the urine sample indicates a poorprognosis of the EBV-associated cancer.
 13. The method according toclaim 12, wherein the poor prognosis comprises low likelihood of overallsurvival or high recurrence for the individual.
 14. The method accordingto claim 12, wherein the EBV associated nucleic acid is DNA.
 15. Themethod according to claim 12, wherein step (b) comprises: (b1)extracting nucleic acid from the urine sample; (b2) amplifying the EBVassociated nucleic acid sequence from the extracted nucleic acid; and(b3) detecting the amplified EBV associated nucleic acid sequence. 16.The method according to claim 12, wherein the EBV associated nucleicacid sequence is derived from at least one fragment of the BamHI-Wregion of the EBV genome.
 17. The method according to claim 16, whereinthe at least one fragment is less than 180 nucleotides.
 18. The methodaccording to claim 12, wherein a polymerase chain reaction (PCR) assay,a reverse transcriptase polymerase chain reaction (RT-PCR) assay, areal-time quantitative polymerase chain reaction (Q-PCR), or a real-timequantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR)assay is performed in step (b).
 19. The method according to claim 18,wherein a pair of primers used in the PCR, RT-PCR, Q-PCR or Q-RT-PCRassay are selected from the group consisting of SEQ ID NO. 1 and SEQ IDNO. 2; and SEQ ID NO. 3 and SEQ ID NO.
 4. 20. The method according toclaim 18, wherein a probe used in the Q-PCR or Q-RT-PCR assay has a DNAsequence of SEQ ID NO. 5 or SEQ ID NO.6.
 21. The method according toclaim 12, wherein the EBV-associated cancer is a nasopharyngealcarcinoma (NPC), a natural-killer-cell lymphoma (NK-lymphoma) or agastric carcinoma.
 22. A method for monitoring an EBV-associated cancerin an individual comprising: (a) obtaining urine samples from theindividual at different time points; and (b) detecting and/orquantifying an EBV associated nucleic acid sequence in the urinesamples; wherein the presence or an increased level of the EBVassociated nucleic acid sequence in the urine samples indicates theprogression of the EBV-associated cancer, and the absence or a decreasedlevel of the EBV associated nucleic acid sequence in the urine samplesindicates the regression of the EBV-associated cancer.
 23. The methodaccording to claim 22 wherein step (b) comprises: (b1) extractingnucleic acid from the urine sample; (b2) amplifying the EBV associatednucleic acid sequence from the extracted nucleic acid; and (b3)detecting and/or quantifying the amplified EBV associated nucleic acidsequence.
 24. The method according to claim 22, wherein step (a) isperformed during the treatment of the EBV-associated cancer, and theabsence or a decreased level of the EBV associated nucleic acid sequencein the urine samples indicates the effectiveness of the treatment. 25.The method according to claim 22, wherein the EBV associated nucleicacid is DNA.
 26. The method according to claim 22, wherein the EBVassociated nucleic acid sequence comprises at least one fragment of theBamHI-W region of the EBV genome.
 27. The method according to claim 26,wherein the at least one fragment is less than 180 nucleotides.
 28. Themethod according to claim 22, wherein a polymerase chain reaction (PCR)assay, a reverse transcriptase polymerase chain reaction (RT-PCR) assay,a real-time quantitative polymerase chain reaction (Q-PCR) assay, or areal-time quantitative reverse transcriptase polymerase chain reaction(Q-RT-PCR) assay is performed in step (b).
 29. The method according toclaim 28, wherein a pair of primers used in the PCR, RT-PCR, Q-PCR orQ-RT-PCR assay are selected from the group consisting of SEQ ID NO. 1and SEQ ID NO. 2; and SEQ ID NO. 3 and SEQ ID NO.
 4. 30. The methodaccording to claim 28, wherein a probe used in the Q-PCR or Q-RT-PCRassay has a DNA sequence of SEQ ID NO. 5 or SEQ ID NO.6.
 31. The methodaccording to claim 22, wherein the EBV-associated cancer is anasopharyngeal carcinoma (NPC), a natural-killer-cell lymphoma(NK-lymphoma) or a gastric carcinoma.
 32. A kit for diagnosis orprognosis of an EBV-associated cancer in an individual comprising: a) afirst unit for extracting nucleic acid from a urine sample from theindividual; and b) a second unit for detecting an EBV associated nucleicacid sequence in the extracted nucleic acid, wherein the second unitcomprises at least one pair of primers for amplifying at least onefragment of the BamHI-W region of the EBV genome.
 33. The kit accordingto claim 32, wherein the EBV associated nucleic acid is DNA.
 34. The kitaccording to claim 32, wherein at least one fragment of the BamHI-Wregion of the EBV genome is less than 180 nucleotides.
 35. The kitaccording to claim 32, wherein the pair of primers selected from thegroup consisting of SEQ ID NO. 1 and SEQ ID NO. 2; and SEQ ID NO. 3 andSEQ ID NO.
 4. 36. The kit according to claim 32, wherein the second unitfurther comprises a probe having a DNA sequence of SEQ ID NO. 5 or SEQID NO.6.
 37. The kit according to claim 32, further comprising a devicefor obtaining a urine sample from an individual.
 38. The kit accordingto claim 32, wherein the EBV-associated cancer is a nasopharyngealcarcinoma (NPC), a natural-killer-cell lymphoma (NK-lymphoma) or agastric carcinoma.
 39. A kit for monitoring an EBV-associated cancer inan individual comprising: a) a plurality of first units for extractingnucleic acid from a urine sample from the individual; and b) a pluralityof second units for detecting and/or quantifying an EBV associatednucleic acid sequence in the extracted nucleic acid, wherein the secondunit comprises at least one pair of primers for amplifying at least onefragment of the BamHI-W region of the EBV genome.
 40. The kit accordingto claim 39, wherein the EBV associated nucleic acid is DNA.
 41. The kitaccording to claim 39, wherein at least one fragment of the BamHI-Wregion of the EBV genome is less than 180 nucleotides.
 42. The kitaccording to claim 39, wherein the at least one pair of primers areselected from the group consisting of SEQ ID NO. 1 and SEQ ID NO. 2; andSEQ ID NO. 3 and SEQ ID NO.
 4. 43. The kit according to claim 39,wherein the second unit further comprises a probe having a DNA sequenceof SEQ ID NO. 5 or SEQ ID NO.6.
 44. The kit according to claim 39,further comprising a device for obtaining a urine sample from anindividual.
 45. The kit according to claim 39, wherein the EBVassociated cancer is a nasopharyngeal carcinoma (NPC), anatural-killer-cell lymphoma (NK-lymphoma) or a gastric carcinoma.